Antibiotic resistance is a major worldwide problem. With excellent efficacy, bioavailability, and minimal toxicity, the frequently prescribed fluoroquinolones are an extremely important drug class. The long-term goals of the Zechiedrich laboratory are to determine how gram-negative bacteria respond to and develop resistance to antimicrobial agents and to use this knowledge to improve diagnosis of antibiotic-resistant bacterial infections, to prolong the usefulness of current drugs, and to aid in the design of new therapeutic modalities. In previous funding periods, we constructed an evolving HIPAA compliant Oracle database that combines patient, hospital, and microbiological data from gram-negative clinical isolates collected since 1999. We discovered that known fluoroquinolone resistance mechanisms account for the resistance phenotype in approximately half of fluoroquinolone-resistant E. coli clinical isolates; therefore, additional unknown resistance mechanisms must exist. To uncover these mechanisms, we developed a unique approach that incorporates genome pooling, sequencing, and SNP analysis. We uncovered extremely problematic novel clinical isolates (resistant to all but two antibiotics and with very high fluoroquinolone MICs). The genomes of these isolates diverged from and could not be mapped to reference genomes. Conserved among all other fluoroquinolone-resistant pools, we found the well-known SNP in the gyrA target gene and three novel SNPs relative to drug-susceptible reference genomes. We also found 283 SNPs in 173 genes associated with fluoroquinolone resistance and resistance to specific additional antimicrobial agents. Relative to a fluoroquinolone-resistant reference genome, we found 35 novel SNPs associated with a fluoroquinolone-susceptible phenotype. The aims of this proposal are Aim 1: Predict how Identified Genetic Variants Alter the Encoded Protein Structure and Function, Aim 2: Define Function of Discovered Genetic Variants, Aim 3: Determine the Molecular Mechanisms of Discovered Protein Variants, and Aim 4: Continue to Discover Genetic Variants Associated with Fluoroquinolone Susceptibility or Fluoroquinolone Resistance Phenotypes. Our team, in the Texas Medical Center in Houston, Texas, is composed of a molecular microbiologist (Zechiedrich), a physician scientist (Hamill), a genomics expert (Sucgang), a computational biologist (Barth), and a crystallographer (Lee). The foundational knowledge gained in achieving these goals is required before new drugs or new targets for new drugs can be identified. A better understanding of how resistance arises will provide vital clues to how it may be avoided. Moreover, the identification of SNPs associated with fluoroquinolone resistance or susceptibility will provide physicians tools to prescribe antibiotics that will be effective, thus saving lives, money, and preserving the current arsenal of antibiotics.